Hi,
I designed Gibson primers (2 primers for fragment and 2 primers for vector) using snapgene.
Then I ran PCR separately for Fragment and Vector.
Fragment DNA was found to be the correct sized PCR product while Vector showed 2 bands instead of 1 band.
Out of those 2 bands I cut the band that was correct sized and tried to elute it out using Qaigen kit but it gave me very less concentration of DNA and had high impurity. I also tried another method to purify DNA using TE buffer overnight incubation at 4C and then used Amicon filters to concentrate the DNA but still concentration of DNA was low and it was impure.
How should I proceed? I would need the amplified DNA vector for Gibson cloning.
Thanks.
1. If you are getting 2 clear bands, that means that your primers are not specific enough and are binding to sites other than the target site.
2. Are you using the correct Qiagen clean up kit? There are 2 types of clean up kits. One is used to purify pcr product without running the pcr product on gel (QIAquick PCR Purification Kit). The other is used to clean up DNA band that is cut from the gel (QIAquick Gel Extraction Kit).
3. If you were using QIAquick Gel Extraction Kit, maybe you can try to recheck the protocol. I've used this kit for a long time and had never encounter any problem before.
It's perfectly normal to lose most of the DNA and have high UV contamination with gel clean-up kits. These almost never work as advertised. If you got any detectable amount of vector back, you can still assemble that. I have routinely assembled vector DNA that looked unworkable -- like 5ng/ul with enormous UV peaks. Just use as much as you can. You just need one colony....
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